Dan Eastwood
Swansea University, Biosciences, Faculty Member
Globalization and international trade have impacted organisms around the world leading to a considerable number of species establishing in new geographic areas. Many organisms have taken advantage of human-made environments, including... more
Globalization and international trade have impacted organisms around the world leading to a considerable number of species establishing in new geographic areas. Many organisms have taken advantage of human-made environments, including buildings. One such species is the dry rot fungus Serpula lacrymans, which is the most aggressive wood-decay fungus in indoor environments in temperate regions. By using population genomic analyses of 36 full genome sequenced isolates, we revealed that isolates from Europe and Japan are highly divergent and that these populations split 3,000 - 19,000 generations ago, probably predating human influence. Approximately 250 generations ago, the European population went through a tight bottleneck, likely corresponding to the time it colonized the built environment. Moreover, evidence of admixture between European and Japanese populations was shown in an isolate from New Zealand. Genomic analyses revealed that low differentiation appeared in genes with funct...
Research Interests: Biology, Ecology, Medicine, Molecular Ecology, Biological Sciences, and 3 morePopulation, Fungus, and Dry Rot
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Dating Wood Rot Specific lineages within the basidiomycete fungi, white rot species, have evolved the ability to break up a major structural component of woody plants, lignin, relative to their non–lignin-decaying brown rot relatives.... more
Dating Wood Rot Specific lineages within the basidiomycete fungi, white rot species, have evolved the ability to break up a major structural component of woody plants, lignin, relative to their non–lignin-decaying brown rot relatives. Through the deep phylogenetic sampling of fungal genomes, Floudas et al. (p. 1715 ; see the Perspective by Hittinger ) mapped the detailed evolution of wood-degrading enzymes. A key peroxidase and other enzymes involved in lignin decay were present in the common ancestor of the Agaricomycetes. These genes then expanded through gene duplications in parallel, giving rise to white rot lineages.
Research Interests: Botany, Phylogenetics, Molecular Evolution, Science, Biology, and 15 moreMedicine, Multidisciplinary, Wood, Molecular Phylogenetics and Evolution, Lignin, Genome, Peroxidases, Fungus, Phylogenetic Tree, Fungal genome size, White Rot, Bayes Theorem, BASIDIOMYCOTA, Lignin Peroxidase, and Agaricomycetes
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ABSTRACT Introduction Basidiomycete species are a key component in wood degradation and have a great influence in carbon cycling. Two major mechanisms of wood decay have been characterized, termed white rot and brown rot. White rot... more
ABSTRACT Introduction Basidiomycete species are a key component in wood degradation and have a great influence in carbon cycling. Two major mechanisms of wood decay have been characterized, termed white rot and brown rot. White rot species effectively degrade lignin, cellulose and hemicellulose while brown rot species degrade mainly the latter two components leaving lignin almost intact (1). The brown rot mechanism has evolved independently in at least six different lineages of Agaricomycotina (2). The genome sequence of the brown rot species Postia placenta revealed that the species has undergone multiple losses in gene families related to wood degradation in comparison to the genome sequence of the white rot species Phanerochaete chrysosporium (3). Recently the genome sequence of Serpula lacrymans, which belongs in an independently evolved brown rot lineage in the Boletales, was produced by JGI and along with the increasing sequenced genomes of other white rot species this provides the significant chance to study the evolution of wood degradation mechanism in the level of gene duplications and losses. Materials and Methods 7 Basidiomycete and 3 Ascomycete genomes were used in the study, using protein sequences published in various studies as queries and performing blastp searches, having as target 19 gene families diversely involved in wood degradation (Table1). The sequences for each gene family were aligned using MAFFT. RAxML analysis was performed for each family, using the appropriate protein evolution model (estimated with ProtTest). The RAxML bootstrapped tree was used as the gene tree along with the species tree shown in Fig. 1 to perform reconciliation analyses using Notung (4). The default costs for duplications (1.5) and losses (1.0) were used. Two different bootstrap values (EWT 90 and 75) were used to define well supported clades on the gene trees in order to perform rearrangements. The calculated duplications and losses for each tree after the rearrangements were eventually mapped on the organismal tree. Conclusions • The common ancestor of the 7 Basdiomycete species was estimated to have from 66 to 83 copies of different CAZY gene copies, depending on the EWT settings employed. The brown rot species S. lacrymans and P. placenta and the mycorrhizal species L. bicolor have undergone extensive gene losses in the CAZY gene families in comparison to the common ancestor of the Basidiomycete species and also in comparison to the white rot species or the soil saprotroph (C. cinerea) in the dataset. The results suggest convergent evolution in the two independently evolved brown rot lineages while the results for L. bicolor are in agreement with the mycorrhizal status of the species, which acquires carbon sources mainly from host-derived photosynthate, not through saprotrophy. • Regarding gene families with roles in oxidoreductative procedures, S. lacrymans and P. placenta again have a reduced number of gene copies in comparison to the rest of the Basidiomycetes or the common Basidiomycete ancestor (which possessed 27-29 copies of these gene families), but in this case this is more attributed not to extensive gene losses in the brown rot species, but mainly to gene duplications in the rest of the species. The suggested duplication events are prominent in the Class II peroxidases for P. chrysosporium and H. annosum and in the multicopper oxidases for H. annosum and C. cinerea.
Efficient lignin depolymerization is unique to the wood decay basidiomycetes, collectively referred to as white rot fungi. Phanerochaete chrysosporium simultaneously degrades lignin and cellulose, whereas the closely related species,... more
Efficient lignin depolymerization is unique to the wood decay basidiomycetes, collectively referred to as white rot fungi. Phanerochaete chrysosporium simultaneously degrades lignin and cellulose, whereas the closely related species, Ceriporiopsis subvermispora, also depolymerizes lignin but may do so with relatively little cellulose degradation. To investigate the basis for selective ligninolysis, we conducted comparative genome analysis of C. subvermispora and P. chrysosporium. Genes encoding manganese peroxidase numbered 13 and five in C. subvermispora and P. chrysosporium, respectively. In addition, the C. subvermispora genome contains at least seven genes predicted to encode laccases, whereas the P. chrysosporium genome contains none. We also observed expansion of the number of C. subvermispora desaturase-encoding genes putatively involved in lipid metabolism. Microarray-based transcriptome analysis showed substantial up-regulation of several desaturase and MnP genes in wood-co...
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Molecular techniques are now routinely used in the identification, detection and analysis of wood degrading organisms. An overview of some of the early work on nucleic acid isolation and characterization will be followed by a discussion... more
Molecular techniques are now routinely used in the identification, detection and analysis of wood degrading organisms. An overview of some of the early work on nucleic acid isolation and characterization will be followed by a discussion of the power of sequencing and other procedures for better understanding: the mechanisms involved in the biological degradation of wood, the metabolic basis of preservative function and ultimately the evolutionary history and ecological function of some of these unique organisms.
2013. International Research Group on Wood Protection, Stockholm, Sweden. IRG Document IRG/WP 13-20528. 9 pp.
2013. International Research Group on Wood Protection, Stockholm, Sweden. IRG Document IRG/WP 13-20528. 9 pp.
Brown rot decay removes cellulose and hemicellulose from wood - residual lignin contributing up to 30% of forest soil carbon - and is derived from an ancestral white rot saprotrophy in which both lignin and cellulose are decomposed.... more
Brown rot decay removes cellulose and hemicellulose from wood - residual lignin contributing up to 30% of forest soil carbon - and is derived from an ancestral white rot saprotrophy in which both lignin and cellulose are decomposed. Comparative and functional genomics of the "dry rot" fungus Serpula lacrymans, derived from forest ancestors, demonstrated that the evolution of both ectomycorrhizal biotrophy and brown rot saprotrophy were accompanied by reductions and losses in specific protein families, suggesting adaptation to an intercellular interaction with plant tissue. Transcriptome and proteome analysis also identified differences in wood decomposition in S. lacrymans relative to the brown rot Postia placenta. Furthermore, fungal nutritional mode diversification suggests that the boreal forest biome originated via genetic coevolution of above- and below-ground biota.
Research Interests: Genetics, Genomics, Coevolution, Science, Symbiosis, and 26 moreFungi, Biodiversity, Multidisciplinary, Functional Genomics, Phylogeny, Wood, Oxidoreductases, Functional diversity, Lignin, Trees, Boreal Forest, Biological evolution, Wood Decay Fungi, Molecular dating, Angiosperms, Peroxidases, Mycorrhizae, Proteome analysis, Plant Cell Wall, S100 protein family, Biota, Cell Wall, Forest Soil Ecology, Proteome, BASIDIOMYCOTA, and Gene expression profiling
Wood is a major pool of organic carbon that is highly resistant to decay, owing largely to the presence of lignin. The only organisms capable of substantial lignin decay are white rot fungi in the Agaricomycetes, which also contains... more
Wood is a major pool of organic carbon that is highly resistant to decay, owing largely to the presence of lignin. The only organisms capable of substantial lignin decay are white rot fungi in the Agaricomycetes, which also contains non–lignin-degrading brown rot and ectomycorrhizal species. Comparative analyses of 31 fungal genomes (12 generated for this study) suggest that lignin-degrading peroxidases expanded in the lineage leading to the ancestor of the Agaricomycetes, which is reconstructed as a white rot species, and then contracted in parallel lineages leading to brown rot and mycorrhizal species. Molecular clock analyses suggest that the origin of lignin degradation might have coincided with the sharp decrease in the rate of organic carbon burial around the end of the Carboniferous period.
Serpula lacrymans, the causative agent of dry rot timber decay in buildings, is a Basidiomycete fungus in the Boletales clade. It owes its destructiveness to a uniquely well-developed capacity to colonize by rapid mycelial spread from... more
Serpula lacrymans, the causative agent of dry rot timber decay in buildings, is a Basidiomycete fungus in the Boletales clade. It owes its destructiveness to a uniquely well-developed capacity to colonize by rapid mycelial spread from sites of initial spore infection, coupled with aggressive degradation of wood cellulose. Genomic methods have recently elucidated the evolution and enzymic repertoire of the fungus, suggesting that it has a distinctive mode of brown rot wood decay. Using novel methods to image nutrient translocation, its mycelium has been modeled as a highly responsive resource-supply network. Dry rot is preventable by keeping timber dry. However, in established outbreaks, further mycelial spread can be arrested by inhibitors of translocation.
Efficient lignin depolymerization is unique to the wood decay basidiomycetes, collectively referred to as white rot fungi. Phanerochaete chrysosporium simultaneously degrades lignin and cellulose, whereas the closely related species,... more
Efficient lignin depolymerization is unique to the wood decay basidiomycetes, collectively referred to as white rot fungi. Phanerochaete chrysosporium simultaneously degrades lignin and cellulose, whereas the closely related species, Ceriporiopsis subvermispora, also depolymerizes lignin but may do so with relatively little cellulose degradation. To investigate the basis for selective ligninolysis, we conducted comparative genome analysis of C. subvermispora and P. chrysosporium. Genes encoding manganese peroxidase numbered 13 and five in C. subvermispora and P. chrysosporium, respectively. In addition, the C. subvermispora genome contains at least seven genes predicted to encode laccases, whereas the P. chrysosporium genome contains none. We also observed expansion of the number of C. subvermispora desaturase-encoding genes putatively involved in lipid metabolism. Microarray-based transcriptome analysis showed substantial upregulation of several desaturase and MnP genes in wood-containing medium. MS identified MnP proteins in C. subvermispora culture filtrates, but none in P. chrysosporium cultures. These results support the importance of MnP and a lignin degradation mechanism whereby cleavage of the dominant nonphenolic structures is mediated by lipid peroxidation products. Two C. subvermispora genes were predicted to encode peroxidases structurally similar to P. chrysosporium lignin peroxidase and, following heterologous expression in Escherichia coli, the enzymes were shown to oxidize high redox potential substrates, but not Mn 2+. Apart from oxidative lignin degradation, we also examined cellulolytic and hemicellulolytic systems in both fungi. In summary, the C. subvermispora genetic inventory and expression patterns exhibit increased oxidoreductase potential and diminished cellulolytic capability relative to P. chrysosporium.
The vast quantities of gene expression profiling data produced in microarray studies, and the more precise quantitative PCR, are often not statistically analysed to their full potential. Previous studies have summarised gene expression... more
The vast quantities of gene expression profiling data produced in microarray studies, and the more precise quantitative PCR, are often not statistically analysed to their full potential. Previous studies have summarised gene expression profiles using simple descriptive statistics, basic analysis of variance (ANOVA) and the clustering of genes based on simple models fitted to their expression profiles over time. We report the novel application of statistical non-linear regression modelling techniques to describe the shapes of expression profiles for the fungus Agaricus bisporus, quantified by PCR, and for E. coli and Rattus norvegicus, using microarray technology. The use of parametric non-linear regression models provides a more precise description of expression profiles, reducing the "noise" of the raw data to produce a clear "signal" given by the fitted curve, and describing each profile with a small number of biologically interpretable parameters. This approach then allows the direct comparison and clustering of the shapes of response patterns between genes and potentially enables a greater exploration and interpretation of the biological processes driving gene expression. Results: Quantitative reverse transcriptase PCR-derived time-course data of genes were modelled. "Split-line" or "broken-stick" regression identified the initial time of gene up-regulation, enabling the classification of genes into those with primary and secondary responses. Five-day profiles were modelled using the biologically-oriented, critical exponential curve, y(t) = A + (B + Ct)Rt + ε. This non-linear regression approach allowed the expression patterns for different genes to be compared in terms of curve shape, time of maximal transcript level and the decline and asymptotic response levels. Three distinct regulatory patterns were identified for the five genes studied. Applying the regression modelling approach to microarray-derived time course data allowed 11% of the Escherichia coli features to be fitted by an exponential function, and 25% of the Rattus norvegicus features could be described by the critical exponential model, all with statistical significance of p < 0.05. Conclusion: The statistical non-linear regression approaches presented in this study provide detailed biologically oriented descriptions of individual gene expression profiles, using biologically variable data to generate a set of defining parameters. These approaches have application to the modelling and greater interpretation of profiles obtained across a wide range of platforms, such as microarrays. Through careful choice of appropriate model forms, such statistical regression approaches allow an improved comparison of gene expression profiles, and may provide an approach for the greater understanding of common regulatory mechanisms between genes.
Research Interests: Kinetics, Statistical Modelling, Gene expression, Quantitative PCR, Escherichia coli, and 16 moreAnimals, Regression Analysis, Statistical Significance, Genes, Optometry and Ophthalmology, Rats, Analysis of Variance, Biological Process, Agaricus, Reverse Transcriptase, Exponential Function, Time Course, Non linear regression, Biochemistry and cell biology, Gene Expression Regulation, and Gene expression profiling
A double-stranded (ds) RNA hairpin-mediated down-regulation system was developed for the cultivated mushroom Agaricus bisporus, and the role of the urea cycle enzyme argininosuccinate lyase (asl) in mushroom post-harvest development was... more
A double-stranded (ds) RNA hairpin-mediated down-regulation system was developed for the cultivated mushroom Agaricus bisporus, and the role of the urea cycle enzyme argininosuccinate lyase (asl) in mushroom post-harvest development was investigated. Hairpin expression vectors were constructed to initiate down-regulation of asl and introduced into A. bisporus by Agrobacterium tumefaciens-mediated transformation. Transcripts of asl were significantly reduced (93.1 and 99.9 %) in two transformants and hairpin vector transgene sequences were maintained throughout sporophore development. Single and multiple hairpin integration events were observed in Southern analysis. Transformants with down-regulated asl exhibited reduced yield and cap expansion during post-harvest sporophore development. There were no detectable differences in urea levels between the hairpin-transformed and control strains. This is the first report of reduced gene expression resulting from the introduction of dsRNA hairpins in A. bisporus and the applications of this technology will facilitate functional studies in the mushroom.
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During the cultivation of mushrooms on straw, the degradation of lignocellulose takes place under the action of enzymes releasing degradation products with small molecular weight, some of which are potentially valuable (sugars, phenolics,... more
During the cultivation of mushrooms on straw, the degradation of lignocellulose takes place under the action of enzymes releasing degradation products with small molecular weight, some of which are potentially valuable (sugars, phenolics, fatty acids, etc). These compounds may be extracted from straw after mushroom cultivation in two stages: an aqueous extraction followed by a solvent extraction. The present work is focused on the first stage of the process where water soluble compounds are extracted. It is important to conduct experiments at a scale that would reflect the amount of waste straw generated by a mushroom farm. A study was performed using a vessel of 300 L capacity with mixing impeller, by observing the influence of the temperature, extraction time and water-to-dry straw ratio on the total extracted matter and especially on sugar and phenolic compounds yields. The optimum extraction conditions were determined by taking into account the yields. From this study, it emerged that the room temperature is recommended, the water-to-straw ratio would not exceed 40:1 kg kg -1 of dry straw and the extraction time should be limited to 4 h
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This work aims to recover lignocellulosic waste in an environmentally friendly process, as an alternative to the energy-intensive technologies: steam explosion, subcritical and/or supercritical water treatment, decomposition through... more
This work aims to recover lignocellulosic waste in an environmentally friendly process, as an alternative to the energy-intensive technologies: steam explosion, subcritical and/or supercritical water treatment, decomposition through pyrolisis, etc. The study assesses the optimum of potentially valuable compounds from straw degraded by the fungus Pleurotus ostreatus. The effects of solvent nature, temperature and extraction time were quantified by material balances with a special view to the extracts obtained. Confirmation of the effectiveness of the operations was done by spectrophotometric, HPLC and LC-MS analyses. Following this study, a farm-based technology was tentatively conceived, requiring neither special skills nor special utilities, to obtain a semi-product for further processing. A centralised technology could be also taken into account to process the straw by direct extraction with hot solvents, in order to obtain products yields three times higher than in the case of the aqueous extraction followed by solvent extraction at 20 °C.
Two spectrophotometric assays have been developed to monitor breakdown of the lignin component of plant lignocellulose: a continuous fluorescent assay involving fluorescently modified lignin, and a UV-vis assay involving chemically... more
Two spectrophotometric assays have been developed to monitor breakdown of the lignin component of plant lignocellulose: a continuous fluorescent assay involving fluorescently modified lignin, and a UV-vis assay involving chemically nitrated lignin. These assays have been used to analyse lignin degradation activity in bacterial and fungal lignin degraders, and to identify additional soil bacteria that show activity for lignin degradation. Two soil bacteria known to act as aromatic degraders, Pseudomonas putida and Rhodococcus sp. RHA1, consistently showed activity in these assays, and these strains were shown in a small scale experiment to breakdown lignocellulose, producing a number of monocyclic phenolic products. Using milled wood lignin prepared from wheat straw, pine, and miscanthus, some bacterial lignin degraders were found to show specificity for lignin type. These assays could be used to identify novel lignin degraders for breakdown of plant lignocellulose.
Metallothioneins are a class of small cysteine-rich proteins that have been associated with increased tolerance to metal and oxidative stresses in animals, plants, and fungi. We investigated a metallothionein-like (mt-like) gene shown... more
Metallothioneins are a class of small cysteine-rich proteins that have been associated with increased tolerance to metal and oxidative stresses in animals, plants, and fungi. We investigated a metallothionein-like (mt-like) gene shown previously to be upregulated in fruiting bodies of the fungus Agaricus bisporus in response to post-harvest storage. Analysis of an A. bisporus genomic DNA cosmid library identified two similar mt-like genes (met1 and met2) arranged as a bidirectional gene pair transcribed from the same promoter region. The promoter contained regulatory elements including 9 metal responsive elements and a CAAT box region 220 bp downstream of met1 that showed striking similarity to a feature in Coprinopsis cinerea mt-like gene promoters. Transcriptional analysis showed that both met genes are significantly and rapidly (within 3 hours) upregulated during post-harvest storage and expression is significantly greater in stipe and cap tissues compared with the gills. However, a strong directionality of the promoter was demonstrated, as transcript levels of met1 were at least two orders of magnitude greater than those of met2 in all samples tested.
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Functional gene studies in the cultivated white button mushroom Agaricus bisporus have been constrained by the absence of effective gene-silencing tools. Using two endogenous genes from A. bisporus, we have tested the utility of dsRNA... more
Functional gene studies in the cultivated white button mushroom Agaricus bisporus have been constrained by the absence of effective gene-silencing tools. Using two endogenous genes from A. bisporus, we have tested the utility of dsRNA hairpin constructs to mediate downregulation of specific genes. Hairpin constructs for genes encoding orotidine 5'-monophosphate decarboxylase (URA3) and carboxin resistance (CBX) were introduced into A. bisporus using Agrobacterium-mediate transfection. Although predicted changes in phenotype were not observed in vitro, quantitative-PCR analyses indicated unambiguously that transcripts in several transformants were substantially reduced compared with the non-transformed controls. Interestingly, some hairpin transformants exhibited increased transcription of target genes. Our observations show that hairpin transgenic sequences can mediate downregulation of A. bisporus endogenous genes and that the technology has the potential to expedite functional genomics of the mushroom.
The enzymic oxidation of the polyunsaturated fatty acid-linoleic acid leads, in fungi, to the formation of a unique class of nonconjugated hydroperoxides, which are cleaved to form eight-carbon volatiles characteristic of mushroom and... more
The enzymic oxidation of the polyunsaturated fatty acid-linoleic acid leads, in fungi, to the formation of a unique class of nonconjugated hydroperoxides, which are cleaved to form eight-carbon volatiles characteristic of mushroom and fungal flavor. However, the enzymes involved in this biosynthetic pathway, the bioavailability of the fatty acid substrate, and the occurrence of the reaction products (hydroperoxides and eight-carbon volatiles) are not fully understood. This study investigated the lipids, fatty acids, and hydroperoxide levels, as well as eight-carbon volatile variations in the fungal model Agaricus bisporus, according to four parameters: sporophore development, postharvest storage, tissue type, and damage. Eight-carbon volatiles were measured using solid phase microextraction and gas chromatography-mass spectrometry. Tissue disruption had a major impact on the volatile profile, both qualitatively and quantitatively; 3-octanone was identified as the main eight-carbon volatile in whole and sliced sporophore, an observation overlooked in previous studies due to the use of tissue disruption and solvent extraction for analysis. Fatty acid oxidation and eight-carbon volatile emissions decreased with sporophore development and storage, and differed according to tissue type. The release of 1-octen-3-ol and 3-octanone by incubation of sporophore tissue homogenate with free linoleic acid was inhibited by acetylsalicylic acid, providing evidence for the involvement of a heme-dioxygenase in eight-carbon volatile production.
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Eight-carbon volatiles are ubiquitous among fungi and characteristic of the fungal aroma. They are the product of the oxidation and cleavage of the fatty acid linoleic acid and are classified as oxylipins, molecules taking part in a wide... more
Eight-carbon volatiles are ubiquitous among fungi and characteristic of the fungal aroma. They are the product of the oxidation and cleavage of the fatty acid linoleic acid and are classified as oxylipins, molecules taking part in a wide range of biological processes. Their involvement in the fungal aroma, interactions with pests and pathogens, and reproductive events are reviewed here, as well as the enzymic systems involved in their biosynthesis.
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The complete oat gene and cDNA from the commercial mushroom, Agaricus bisporus, encoding ornithine aminotransferase (OAT) was characterized. The gene encodes a 466 amino acid protein and provides the first fully reported homobasidiomycete... more
The complete oat gene and cDNA from the commercial mushroom, Agaricus bisporus, encoding ornithine aminotransferase (OAT) was characterized. The gene encodes a 466 amino acid protein and provides the first fully reported homobasidiomycete OAT protein sequence. The gene is interrupted by ten introns, and no mitochondrial targeting motif was present pointing to a cytoplasmic localization. The function of the gene was demonstrated by complementation of a Saccharomyces cerevisiae mutant unable to utilize ornithine as a sole source of nitrogen with an A. bisporus oat cDNA construct. Northern analysis of the oat gene together with the pruA gene (encoding Δ1-pyrroline-5-carboxylate dehydrogenase) showed that transcripts of both genes were lower during the first stages of fruiting body development. The higher expression of the oat gene in later stages of development, suggests the importance of ornithine metabolism for the redistribution of metabolites in the developing mushroom. Hplc analysis of all amino acids revealed that ornithine levels increased during fruiting body development whereas proline levels fell.
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Cloning and characterisation of a putative riboflavin-aldehyde-forming enzyme gene (raf) from the cultivated mushroom Agaricus bisporus and its expression during morphogenesis are described. Three cDNA clones were isolated following... more
Cloning and characterisation of a putative riboflavin-aldehyde-forming enzyme gene (raf) from the cultivated mushroom Agaricus bisporus and its expression during morphogenesis are described. Three cDNA clones were isolated following differential screening of cDNA libraries from rapidly expanding sporophores and post-harvest stored sporophores. The cDNA sequence and predicted translation analysis revealed an open reading frame (ORF) of 348 nucleotides encoding a polypeptide of 115 amino acids, with three introns (56-66 bases) interrupting the genomic ORF. Blast X searches of the databases with the gene sequence showed homology (40% identity and 56% similarity) to the riboflavin-aldehyde-forming enzyme gene from Schizophyllum commune. In A.bisporus, the raf gene sequence upstream of the ORF contained a large CT-rich putative regulatory element (-64 to -24 bases) found in highly expressed genes in various mushrooms, and a 6-base motif present in the 3′ end of the genomic sequence, but not in the corresponding 3′ non-coding part of the cDNA, was identified. The raf gene transcripts increased abundantly in rapidly developing sporophores as well in post-harvest stored sporophores. Differential expression of the raf gene transcripts in different tissues of the sporophore was also observed, with higher levels in the stipe compared with the cap and gills. The temporal and spatial expression patterns observed suggest transcriptional regulation of the raf gene during A. bisporus morphogenesis.
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Fruit body initials of Agaricus bisporus contain high levels of urea, which decrease in the following developmental stages until stage 4 (harvest) when urea levels increase again. At storage, the high urea content may affect the quality... more
Fruit body initials of Agaricus bisporus contain high levels of urea, which decrease in the following developmental stages until stage 4 (harvest) when urea levels increase again. At storage, the high urea content may affect the quality of the mushroom, i.e. by the formation of ammonia from urea through the action of urease (EC 3.5.1.5). Despite the abundance of urea in the edible mushroom A. bisporus, little is known about its physiological role. The urease gene of A. bisporus and its promoter region were identified and cloned. The coding part of the genomic DNA was interrupted by nine introns as confirmed by cDNA analysis. The first full homobasidiomycete urease protein sequence obtained comprised 838 amino acids (molecular mass 90,694 Da, pI 5.8). An alignment with fungal, plant and bacterial ureases revealed a high conservation. The expression of the urease gene, measured by Northern analyses, was studied both during normal development of fruit bodies and during post-harvest senescence. Expression in normal development was significantly up-regulated in developmental stages 5 and 6. During post-harvest senescence, the expression of urease was mainly observed in the stipe tissue; expression decreased on the first day and remained at a basal level through the remaining sampling period.
Research Interests: Applied microbiology, Multidisciplinary, Phylogeny, Sequence alignment, Polymerase Chain Reaction, and 10 moreApplied Microbiology and Biotechnology, Molecular cloning, Protein Sequence Analysis, Genomic DNA, Agaricus, Amino Acid Profile, Molecular Mass, Amino Acid Sequence, Base Sequence, and Urease
We screened a cDNA library generated from harvested and stored sporophores of Agaricus bisporus and identified 19 genes with higher transcript levels than at the time of harvest. Five of these genes had no detectable mRNA levels prior to... more
We screened a cDNA library generated from harvested and stored sporophores of Agaricus bisporus and identified 19 genes with higher transcript levels than at the time of harvest. Five of these genes had no detectable mRNA levels prior to detachment from the mycelium. Sequence analysis of ten clones revealed significant similarities to known genes, these code for proteins involved in polymer breakdown and metabolism, cell wall synthesis, stress tolerance, cytochrome P450 activity and DNA binding. The diversity of functions of these genes suggests the changes in the sporophore after harvest involve several different physiological processes.
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Accumulation of high quantities of urea in fruiting bodies is a known feature of larger basidiomycetes. Argininosuccinate synthetase (ASS) and argininosuccinate lyase (ASL) are two ornithine cycle enzymes catalysing the last two steps in... more
Accumulation of high quantities of urea in fruiting bodies is a known feature of larger basidiomycetes. Argininosuccinate synthetase (ASS) and argininosuccinate lyase (ASL) are two ornithine cycle enzymes catalysing the last two steps in the arginine biosynthetic pathway. Arginine is the main precursor for urea formation. In this work the nucleotide sequences of the genes and corresponding cDNAs encoding argininosuccinate synthetase (ass) and argininosuccinate lyase (asl) from Agaricus bisporus were determined. Eight and six introns were present in the ass and asl gene, respectively. The location of four introns in the asl gene were conserved among vertebrate asl genes. Deduced amino acid sequences, representing the first homobasidiomycete ASS and ASL protein sequences, were analysed and compared with their counterparts in other organisms. The ass ORF encoded for a protein of 425 amino acids with a calculated molecular mass of 47 266 Da. An alignment with ASS proteins from other organisms revealed high similarity with fungal and mammalian ASS proteins, 61-63 % and 51-55 % identity, respectively. The asl open reading frame (ORF) encoded a protein of 464 amino acids with an calculated mass of 52 337 Da and similar to ASS shared the highest similarity with fungal ASL proteins, 59-60 % identity. Northern analyses of ass and asl during fruiting body formation and post-harvest development revealed that expression was significantly up-regulated from developmental stage 3 on for all the tissues studied. The expression reached a maximum at the later stages of fruiting body growth, stages 6 and 7. Both ass and asl genes were up-regulated within 3 h after harvest showing that the induction mechanism is very sensitive to the harvest event and emphasizes the importance of the arginine biosynthetic pathway/ornithine cycle in post-harvest physiology.
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Increases in both the levels and the activity of serine proteinase have been previously described in the senescing mushroom Agaricus bisporus. cDNA encoding serine proteinase was amplified by reverse transcriptase-polymerase chain... more
Increases in both the levels and the activity of serine proteinase have been previously described in the senescing mushroom Agaricus bisporus. cDNA encoding serine proteinase was amplified by reverse transcriptase-polymerase chain reaction using a degenerate primer based on the N-terminal sequence of a previously isolated A. bisporus serine proteinase and then cloned. The cDNA was sequenced and shown to be homologous to those of other fungal serine proteinases. Northern analysis showed that this serine proteinase gene (Spr1) was not expressed in freshly harvested sporophores but was strongly up-regulated postharvest and found almost entirely in the stipe of the sporophore (approximately 0.08% of mRNAs 2 days after harvest). Low-level expression was detectable in the flesh (pileus trama) and gill (lamellae) tissues of the cap, but none was detected in the skin (pilei pellis). In three of the cloned cDNAs, sequence analysis showed that the poly(A) tail starts at different positions. Expression of Spr1 in Escherichia coli caused restricted colony growth.
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Our aim was to generate and prove the concept of "smart" plants to monitor plant phosphorus (P) status in Arabidopsis. Smart plants can be genetically engineered by transformation with a construct containing the promoter of a gene... more
Our aim was to generate and prove the concept of "smart" plants to monitor plant phosphorus (P) status in Arabidopsis. Smart plants can be genetically engineered by transformation with a construct containing the promoter of a gene up-regulated specifically by P starvation in an accessible tissue upstream of a marker gene such as β-glucuronidase (GUS). First, using microarrays, we identified genes whose expression changed more than 2.5-fold in shoots of plants growing hydroponically when P, but not N or K, was withheld from the nutrient solution. The transient changes in gene expression occurring immediately (4 h) after P withdrawal were highly variable, and many nonspecific, shock-induced genes were up-regulated during this period. However, two common putative cis-regulatory elements (a PHO-like element and a TATA box-like element) were present significantly more often in the promoters of genes whose expression increased 4 h after the withdrawal of P compared with their general occurrence in the promoters of all genes represented on the microarray. Surprisingly, the expression of only four genes differed between shoots of P-starved and -replete plants 28 h after P was withdrawn. This lull in differential gene expression preceded the differential expression of a new group of 61 genes 100 h after withdrawing P. A literature survey indicated that the expression of many of these "late" genes responded specifically to P starvation. Shoots had reduced P after 100 h, but growth was unaffected. The expression of SQD1, a gene involved in the synthesis of sulfolipids, responded specifically to P starvation and was increased 100 h after withdrawing P. Leaves of Arabidopsis bearing a SQD1::GUS construct showed increased GUS activity after P withdrawal, which was detectable before P starvation limited growth. Hence, smart plants can monitor plant P status. Transferring this technology to crops would allow precision management of P fertilization, thereby maintaining yields while reducing costs, conserving natural resources, and preventing pollution.
Research Interests: Gene expression, Genetic Engineering, Biological Sciences, Phosphorus, Plant Physiology, and 13 morePlant, Polymerase Chain Reaction, Glucuronidase, Arabidopsis, Trace Elements, Phosphates, Natural Resource, Base Sequence, Genetic Markers, Nutrient Solution, Literature survey, Differential Gene Expression, and Differential expression
Double-stranded RNA (dsRNA) elements are routinely associated with the MVX disease of Agaricus bisporus. MVX comprises a wide range of symptoms including; bare cropping areas on commercial beds (primordia disruption), crop delay,... more
Double-stranded RNA (dsRNA) elements are routinely associated with the MVX disease of Agaricus bisporus. MVX comprises a wide range of symptoms including; bare cropping areas on commercial beds (primordia disruption), crop delay, premature veil opening, off- or brown-colored mushrooms, sporophore malformations and loss of crop yield and/or product quality. The 26 MVX dsRNA elements range between 640 bp and 20.2
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... 2000 Balkema, Rotterdam, ISBN 90 5809 143 0 The science of mushroom quality K. Burton, S. Sreenivasaprasad, D. Eastwood, T. Rama ... For example, the expectation of high quality mushrooms,Agaricus bisporus, has been determined by... more
... 2000 Balkema, Rotterdam, ISBN 90 5809 143 0 The science of mushroom quality K. Burton, S. Sreenivasaprasad, D. Eastwood, T. Rama ... For example, the expectation of high quality mushrooms,Agaricus bisporus, has been determined by consumer surveys to be mushrooms of ...
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Author; COMBET E.(Univ. Warwick, Wellesbourne, Gbr) COMBET E.(Coventry Univ., Coventry, Gbr) COMBET E.(Gardiner Inst., Glasgow, Gbr) EASTWOOD DC (Univ. Warwick, Wellesbourne, Gbr) HENDERSON J.(Coventry Univ., Coventry, Gbr) BURTON KS... more
Author; COMBET E.(Univ. Warwick, Wellesbourne, Gbr) COMBET E.(Coventry Univ., Coventry, Gbr) COMBET E.(Gardiner Inst., Glasgow, Gbr) EASTWOOD DC (Univ. Warwick, Wellesbourne, Gbr) HENDERSON J.(Coventry Univ., Coventry, Gbr) BURTON KS (Univ. ...
Fungi play a key role in ecosystem nutrient cycles by scavenging, concentrating, translocating and redistributing nitrogen. To quantify and predict fungal nitrogen redistribution, and assess the importance of the integrity of fungal... more
Fungi play a key role in ecosystem nutrient cycles by scavenging, concentrating, translocating and redistributing nitrogen. To quantify and predict fungal nitrogen redistribution, and assess the importance of the integrity of fungal networks in soil for ecosystem function, we need better understanding of the structures and processes involved. Until recently nitrogen translocation has been experimentally intractable owing to the lack of a suitable radioisotope tracer for nitrogen, and the impossibility of observing nitrogen translocation in real time under realistic conditions. We have developed an imaging method for recording the magnitude and direction of amino acid flow through the whole mycelial network as it captures, assimilates and channels its carbon and nitrogen resources, while growing in realistically heterogeneous soil microcosms. Computer analysis and modeling, based on these digitized video records, can reveal patterns in transport that suggest experimentally testable hypotheses. Experimental approaches that we are developing include genomics and stable isotope NMR to investigate where in the system nitrogen compounds are being acquired and stored, and where they are mobilized for transport or broken down. The results are elucidating the interplay between environment, metabolism, and the development and function of transport networks as mycelium forages in soil. The highly adapted and selected foraging networks of fungi may illuminate fundamental principles applicable to other supply networks.